The goal of this work is to understand the mechanisms by which a nascent peptide encoded by an upstream open reading frame (uORF) controls the movement of ribosomes on mRNA and regulates gene expression. The arginine attenuator peptide (AAP) is encoded by a uORF in the 5'-leader regions of mRNAs specifying a fungal arginine (Arg) biosynthetic enzyme; it reduces gene expression in response to Arg. AAP-mediated regulation is observed in vivo in both Neurospora crassa and Saccharomyces cerevisiae and in vitro, using fungal, plant and animal extracts. The nascent AAP and Arg cause the ribosome to stall. When the AAP functions as a uORF, the ribosome stalls at the termination step. The stalled ribosomes block scanning ribosomes, decreasing gene expression by reducing ribosomal access to the downstream reading frame. The AAP also functions as an internal polypeptide domain to stall ribosomes involved in elongation. Our data leads to a regulatory model in which the AAP adopts a conformation in the ribosome that, with Arg, interferes with decoding at the A-site, or with another step crucial for elongation or termination. A second aspect of regulation by the AAP is that it controls mRNA stability. Both S. cerevisiae CPA1 and N. crassa arg-2 mRNA levels are affected by nonsense-mediated mRNA decay (NMD). AAP-mediated stalling promotes NMD of the CPA1 mRNA. In the absence of AAP-mediated stalling, NMD of the CPA1 mRNA can be promoted by increased ribosome occupancy of the uORF. This data supports a regulatory model in which ribosome stalling at the uORF termination codon in response to Arg destabilizes CPA1 mRNA by increasing the extent of nonsense codon recognition by NMD. This link between AAP-mediated stalling and NMD provides unique opportunities for assessing the cis- and trans-acting elements that contribute to NMD. N. crassa, like many fungi of medical, agricultural, and economic importance, but unlike S. cerevisiae, has clear homologs of elF4AIII, Y14, and Magoh, exon junction complex proteins which are involved in engaging NMD in mammals and are implicated in activating the translation of mRNAs with which they associate. Specific aims are as follows: 1. Elucidate the mechanism of ribosome stalling by analyzing the structure of the AAP through (a) cross-linking the nascent AAP to the ribosome to examine their interactions (b) assessing the conformation of the AAP in the ribosome by comparing it to other nascent chains with known conformations; (c) directly examining AAP structure and how Arg affects it by 2D-NMR. 2. Exploit the regulated expression of yeast CPA1 to test key aspects of the faux-UTR model of NMD and follow up on novel observations that AAP- mediated ribosome stalling triggers NMD. 3. Use new and existing N. crassa strains to determine the functions of Neurospora NMD and EJC factors in translation and mRNA metabolism through the analysis of the phenotypes that result from the loss of these functions. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]